TISSUE CULTURE OF PERSOONIA SPECIES FOR HORTICULTURE AND RESTORATION

Full text

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This is the peer reviewed version of: Offord, C.A., Rollason, A. and Frith, A. (2015).
Tissue culture of Persoonia species for horticulture and restoration. Acta Hortic. 1101,
75-80 DOI: 10.17660/ActaHortic.2015.1097.17 which has been published in final
form at
http://dx.doi.org/10.17660/ActaHortic.2015.1097.17

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Tissue culture of Persoonia species for horticulture and restoration
Catherine A. Offord, Amanda Rollason, Allison Frith
The Australian PlantBank, the Australian Botanic Garden, Mount Annan, NSW 2567,
Australia
Keywords
Persoonia, Proteaceae, tissue culture, seed germination, ornamental, restoration,
conservation
Abstract
Persoonia (Proteaceae) is an Australian endemic genus of Proteaceae many
species of which are horticulturally desirable. A large number of species are
endangered in the wild and the restoration industry is interested in effective
ways to propagate them. While high volume regeneration of persoonias from
seeds and cuttings remains problematic, tissue culture may be an alternative or
complementary technique. Further work on this taxon is required to optimise
multiplication of cultures and the transition of explants to the ex vitro
environment.
INTRODUCTION
Persoonia is an endemic Australian genus of Proteaceae known as geebungs
in eastern Australia and snottygobbles in the west. There are over 100 species,
generally characterised by their shrubby habit and small but attractive yellow flowers.
They are underexploited horticulturally, despite their desirability. There are several
species with prostrate habits which makes them excellent rockery plants (Fig. 1),
groundcovers or hanging plants. A small number of persoonias are used in the
domestic market for their foliage and flowers, but are primarily bush-harvested.
A large number of eastern Australian persoonias are highly threatened and are
the subject of conservation in situ and ex situ. The restoration industry also desires to
include persoonias in replanting programs but is hampered by the lack of suitable
material and lack of knowledge of their biology.
In general, the cultivation of persoonias is limited by difficulty with
propagation. The fruit is a drupe that has fleshy a mesocarp surrounding a stony
endocarp which constrains the embryo that consists of 2 or more cotyledons.
Additionally, they may demonstrate physiological dormancy that may be overcome
by treatment with GA3 and smoke, once the mesocarp is removed and the endocarp is
scarified or removed (Mullins et al., 2002). Propagation by seeds is therefore difficult

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(Offord et al., 2012) and vegetative propagation often slow or impossible (Weston,
2003). Preliminary work on tissue culture of a range of eastern Australian persoonia
species offers potential for propagation by tissue culture.
MATERIALS AND METHODS
Plant Material
Plant material, fruits or cuttings, was originally collected from wild
populations and accessioned on the Royal Botanic Gardens EMu database (Table 1).
Stock plants established from cuttings were grown in a shadehouse under 50% shade
in a coir/sand mix with added nutrients (Offord et al. 1998) with daily watering.
Fruits were cleaned of the fleshy mesocarp, dried at 15% RH and 15ºC and
then stored in hermetically sealed Aluminium envelope at 5 ºC until used or used
fresh.
Initiation
Vegetative material. Excised shoots were washed under running water for 1
hr, sterilised in 1% sodium hypochlorite for 15 min and rinsed 3 times in sterile water.
The leaves were minimised and shoots cut to a length of 7-15 mm and placed onto a
modified Murashige and Skoog (1962) medium with the macro and micro nutrient
halved (HMS), with 30 g l-1 sucrose and 8 gl-1 agar, at pH 5.75. They were incubated
at 23 ºC + 2 ºC under 50 µmol m-2 s-1 photosynthetically active photon flux density in
a growth room for 16/8 hr dark/light in the diurnal period.
Embryos. Preliminary trials showed that under laboratory conditions, only
embryos released from the endocarps germinated. Decontamination of embryos was
examined by soaking defleshed endocarps/embryo in 4% sodium hypochlorite (with a
drop of Tween 20) for 15 min and cracking the sterile H20-rinsed endocarps in a vice
in a laminar cabinet. Embryos were extracted from the endocarp with forceps (Fig. 2)
and then either placed on sterile agar or subjected to 1% sodium hypochlorite for 5
min, rinsed, and then placed onto sterile agar. They were incubated under the same
conditions as the vegetative material. The embryos were monitored every 2 days and
germination success recorded as the production of both green cotyledons and radicle
>2mm with no observable contamination.
Establishment and shoot multiplication

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Once clean material was obtained, shoots were placed onto multiplication
medium of HMS and 0.2 or 0.5 µM benzyl adenine (BA). Shoots were subcultured
every 6-8 weeks and incubated under the same conditions as for Initiation.
Plant establishment
Twenty explants each of P. linearis, P. pauciflora, P. pinifolia and P. nutans
and were prepared for exflasking by culturing on HMS as before with the addition of
6 µM IBA. After 28 days incubation, the microplants were transferred to a seed
raising tray containing mix consisting of perlite, peat, moss (7:2:1) with 2 g L-1 slow
release fertiliser (Nutricote 12-5.2-10) with 0.64 g L-1 slow release micronutrients
(Micromax), 0.24 g L-1 FeSO4 and 0.2 g L-1 dolomite. The trays were kept at >98%
relative humidity by enclosing them with transparent plastic cloches. The trays were
placed on a bench in a 25% ambient sunlight glasshouse maintained at 22ºC + 2 ºC
with air conditioning and soil temp 2 ºC higher with bottom heating. Vents on the
cloches were opened after 14 days to decease humidity.
RESULTS
Of the cultures successfully established, the majority were from embryo
explants (Table 1). Microbial contamination was the most common cause of failure of
vegetative explants. Only species or hybrids with prostrate habits, P. chamaepitys and
P. recedens x myrtilloides survived initiation using vegetative material and
established sufficiently to be repeatedly subcultured. The growth of both accessions
was vigorous once established.
It was generally easier to obtain clean initiates from embryo cultures than from
vegetative material. Virtually no uncontaminated material arose from fruit/embryos
that were not double sterilised (Treatment 1), whereas sterilisation of the defleshed
endocarp followed by light sterilisation (1% sodium hypochlorite for 5
min)(Treatment 2) gave a good rate of clean, germinated seedlings ie. P. pinifolia 3%
and 16%, and P. linearis 0% and 20%, for the respective treatments. Some species,
such as P. amaliae, yielded high rates of clean material regardless of treatment, ie
53% and 60%. The major problem observed with seed germination was the low
germination percentage, regardless of whether seed was contaminated. Three
‘germination’ types were observed: 1. cotyledons only, 2. cotyledons and radicle, no
further development 3. full conversion to apparently functional seedling (Fig. 3). Only
the latter were used for subsequent culture as they were deemed to be the most
vigorous.

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Once established, explants grew sufficiently to be subcultured every 6-8
weeks with many being cultured to this point for 19 subcultures without apparent
changes to morphology. Explants of all species established in culture had
multiplication rates greater than triple.
Plant Establishment
Only 1 plantlet each of P. pauciflora and P. linearis produced roots ex vitro
and both plantlets fully established as plants and were planted in a common garden
environment. These planst exhibited extremely vigorous growth when compared to
other plants in the same garden that had been grown from cuttings.
DISCUSSION
In general, persoonias appear to be amenable to the tissue culture process but
the choice of explant material is critical. As with many other taxa, juvenile explants,
in this case embryos, were relatively easier to decontaminate and establish in culture.
Of the attempted initiations, only Persoonia chamaepitys and P. recedens x
myrtilloides established from axillary shoot explants. In the majority of cases, the use
of embryo explants resulted in successful establishment. Similar results were
observed in the establishment of P. gunnii (Gorst, 1992) and P. muelleri (Cambecedes
and Balmer, 1995), where apical and axillary shoots were difficult to decontaminate
and turned black and died, while embryo explants were relatively easier to clean and
grew on. Kettlehorn et al. (1996) also successfully used embryos to establish tissue
cultures of P. virgata and P. sericea.
The use of embryos may not be desirable for the promulgation of horticultural
selections but may be useful in production of plants for habitat restoration and
translocation of threatened species, where it is desirable to have as many genotypes as
possible. Further work is required to establish horticultural selections, although it is
encouraging that the highly ornamental prostrate species, P. chamaepitys, was easily
established in culture and maintained vigour for more than 19 subcultures, which may
be related to the general health and vigour of the selection used. Indeed, once
established in culture, we have been able to maintain most persoonia species in a
vigorous state, with the exception of the extremely rare P. nutans. The single attempt
we made to convert explants to fully functioning plants, and the general failure of
microcuttings to strike roots, points to the need for further experimentation to increase
plant production rates.

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Literature cited
Cambecedes, J and Balmer, J. (1995). Lomatia tasmanica and Persoonia muelleri
propagation and commercial horticulture. Final report to the Australian Flora
Foundation http://www.aff.org.au/Balmer_Lomantia_final.pdf accessed 12 May
2015.
Gorst, J.R. (1992) Micropropagation of threatened species of Persoonia. Final report
to the Australian Flora Foundation
http://www.aff.org.au/AFF2_Gorst&Persoonia.htm accessed 12 May 2015.
Kettlehorn, L.M., Johnston, M.E. and Cage, J. (1996). Persoonia virgata, Family
Proteaceae. In ‘Horticulture of Australian Plants’, (Eds M. Burchett and K. Johnston).
pp. 212-217. (UNSW Press: Sydney).
Mullins, G., Koch, J.M.,and Ward, S.C. 2002. Practical method of germination for a
key Jarrah forest species: Snottygobble (Persoonia longifolia). Ecol. Restoration &
Management 3: 97-103.
Offord, C.A., Muir, S and Tyler, J. (1998). Growth of selected Australian plants in
soilless media using coir as a substitute for peat. Australian Journal of Experimental
Agriculture. 38: 879-87.
Offord, C.A., Seed, L.U. and Martyn, A.J. (2012). Perspectives on seed biology of
waratahs (Telopea speciosissima R.Br.) and other Australian Proteaceae. Acta Hort. 937:
801-810.
Weston, P. H. (2003). Proteaceae subfamily Persoonioideae. Australian Plants 22: 62-
78.
Table 1. Species of Persoonia initiated into tissue culture, includes NSW conservation
status Royal Botanic Gardens accession number. Vegetative = vegetative material
taken from cultivated plants, Embryo = embryo released from the surrounding fruit
structures.
Persoonia species
Conservation
status NSW
Accession
No(s)
Type of material
Cultures established
beyond 2 subcultures
P. amaliae
900034
Embryo - fresh
Sub 19
P. bargoensis
Threatened
893066
Vegetative
-
P. chamaepitys
920492
Vegetative
Sub 10
P. curvifolia
900048
Vegetative
-
P. cuspidifera
877377
Vegetative
-
P. linearis
20091801
Embryo - fresh
Sub 19
P. marginata
20070776
932198
Vegetative
-
P. myrtilloides
20091829
Embryo - fresh
Sub 19
P. pauciflora
Threatened
20091917
Embryo - fresh
Sub 19
P. pinifolia
20091891
Vegetative
Sub 19
P. nutans
Threatened
912745
Embryo - fresh
Sub 19
P. recedens x
myrtilloides
920493
Vegetative
Sub 10

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Fig 1. Persoonia chamaepitys has a prostrate habit and is grown as a rockery plant the
Blue Mountains Botanic Garden, Mount Tomah.
Fig 2. Extraction of the non-endospermic embryo of Persoonia pauciflora from the
endocarp.
A B
Fig. 3. Germination of Persoonia embryos: A. cotyledons only, no radicle developed
B. Cotyledons and radicle developed, true leaves emerging.